On-line Solid-phase Extraction Research Articles

How Wastewater Reflects Human Metabolism─Suspect Screening of Pharmaceutical Metabolites in Wastewater Influent.

Pharmaceuticals and their human metabolites are contaminants of emerging concern in the aquatic environment. Most monitoring studies focus on a limited set of parent compounds and even fewer metabolites. However, more than 50% of the most consumed pharmaceuticals are excreted in higher amounts as metabolites than as parents, as confirmed by a literature analysis within this study. Hence, we applied a wide-scope suspect screening approach to identify human pharmaceutical metabolites in wastewater influent from three Swiss treatment plants. Based on consumption amounts and human metabolism data, a suspect list comprising 268 parent compounds and over 1500 metabolites was compiled. Online solid phase extraction combined with liquid chromatography coupled to high-resolution tandem mass spectrometry was used to analyze the samples. Data processing, annotation, and structure elucidation were achieved with various tools, including molecular networking as well as SIRIUS/CSI:FingerID and MetFrag for MS2 spectra rationalization. We confirmed 37 metabolites with reference standards and 16 by human liver S9 incubation experiments. More than 25 metabolites were detected for the first time in influent wastewater. Semiquantification with MS2Quant showed that metabolite to parent concentration ratios were generally lower compared to literature expectations, probably due to further metabolite transformation in the sewer system or limitations in the metabolite detection. Nonetheless, metabolites pose a large fraction to the total pharmaceutical contribution in wastewater, highlighting the need for metabolite inclusion in chemical risk assessment.

Restricted-Access Media Column Switching Online Solid-Phase Extraction UHPLC-MS/MS for the Determination of Seven Type B Trichothecenes in Whole-Grain Preprocessed Foods and Human Exposure Risk Assessment.

With increasing health awareness and the accelerating pace of life, whole-grain prepared foods have gained popularity due to their health benefits and convenience. However, the potential risk of type B trichothecene toxins has also increased, and these mycotoxins in such foods are rarely regulated. In this study, a quantitative method combining a single-valve dual-column automatic online solid-phase extraction system with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed for the first time using restricted-access media columns. This method can simultaneously determine trace residues of seven type B trichothecenes within 15 min. The method is convenient, sensitive (limit of detection and quantification of 0.05-0.6 μg/kg and 0.15-2 μg/kg, respectively), accurate (recovery rates of 90.3%-106.6%, relative standard deviation < 4.3%), and robust (>1000 times). The established method was applied to 160 prepared food samples of eight categories sold in China. At least one toxin was detected in 70% of the samples. Whole-wheat dumpling wrappers had the highest contamination rate (95%) and the highest total content of type B trichothecenes in a single sample (2077.3 μg/kg). Exposure risk assessment indicated that the contamination of whole-grain prepared foods has been underestimated. The total health risk index of whole-wheat dumpling wrappers, which are susceptible to deoxynivalenol, reached 136.41%, posing a significant threat to human health. Effective measures urgently need to be taken to control this risk.

Determination of Ambroxol Levels in Plasma and Cerebrospinal Fluid by Online Solid-Phase Extraction Coupled with Liquid Chromatography-Tandem Mass Spectrometry in GBA-Parkinson Disease Patients

Determination of Ambroxol Levels in Plasma and Cerebrospinal Fluid by Online Solid-Phase Extraction Coupled with Liquid Chromatography-Tandem Mass Spectrometry in GBA-Parkinson Disease Patients

Target and Nontarget Screening to Support Capacity Scaling for Substance Use Assessment through a Statewide Wastewater Surveillance Network in New York.

Wastewater-based epidemiology (WBE) has been widely implemented around the world as a complementary tool to conventional surveillance techniques to inform and improve public health responses. Currently, wastewater surveillance programs in the U.S. are evaluating integrated approaches to address public health challenges across multiple domains, including substance abuse. In this work, we demonstrated the potential of online solid-phase extraction coupled with liquid chromatography-high-resolution mass spectrometry to support targeted quantification and nontargeted analysis of psychoactive and lifestyle substances as a step toward understanding the operational feasibility of a statewide wastewater surveillance program for substance use assessment in New York. Target screening confirmed 39 substances in influent samples collected from 10 wastewater treatment plants with varying sewershed characteristics and is anticipated to meet the throughput demands as the statewide program scales up to full capacity. Nontarget screening prioritized additional compounds for identification at three confidence levels, including psychoactive substances, such as opioid analgesics, phenethylamines, and cathinone derivatives. Consumption rates of 12 target substances detected in over 80% of wastewater samples were similar to those reported by previous U.S.-based WBE studies despite the uncertainty associated with back-calculations. For selected substances, the relative bias in consumption estimates was sensitive to variations in monitoring frequency, and factors beyond human excretion (e.g., as indicated by the parent-to-metabolite ratios) might also contribute to their prevalence at the sewershed scale. Overall, our study marks the initial phase of refining analytical workflows and data interpretation in preparation for the incorporation of substance use assessment into the statewide wastewater surveillance program in New York.

Occurrence and prevalence of per- and polyfluoroalkyl substances in the sediment pore water of mariculture sites: Novel findings of PFASs from the Bohai and Yellow Seas using a newly established analytical method

A new method for the determination of 26 legacy and emerging per- and polyfluoroalkyl substances (PFASs) in marine sediment pore water was developed using online solid phase extraction coupled with liquid chromatography-tandem mass spectrometry. The proposed method requires only about 1 mL of pore water samples. Satisfactory recoveries of most target PFASs (83.55–125.30 %) were achieved, with good precision (RSD of 1.09–16.53 %), linearity (R2 ≥ 0.990), and sensitivity (MDLs: 0.05 ng/L–5.00 ng/L for most PFASs). Subsequently, the method was applied to determine PFASs in the sediment pore water of five mariculture bays in the Bohai and Yellow Seas of China for the first time. Fifteen PFASs were detected with total concentrations ranging from 150.23 ng/L to 1838.48 ng/L (mean = 636.80 ng/L). The ∑PFASs and PFOA concentrations in sediment pore water were remarkably higher than those in surface seawater (tens of ng/L), indicating that the potential toxic effect of PFASs on benthic organisms may be underestimated. PFPeA was mainly distributed in pore water, and the partition of PFHpA (50.99 %) and PFOA (49.01 %) was almost equal in the solid and liquid phases. The proportions of all other PFASs partitioned in marine sediments were significantly higher than those in pore water.

Spherical fluorinated covalent organic polymer for highly efficient and selective extraction of fipronil and its metabolites in soil

Covalent organic polymers (COPs) have garnered considerable attention as promising adsorbents of online solid phase extraction (online SPE). Morphology modulation provides an appealing solution to enhance adsorption efficiency and reduce back-pressure in the absorbent. However, the synthesis of COPs with regular geometric shapes and specific adsorption selectivity remains challenging. In this study, a uniform spherical fluorinated COP (F-sCOP, average diameter: 2.14 μm) was successfully synthesized by Schiff base reaction of 1,3,5-triformylphoroglucinol (TP) and 2,2’-bis(trifluoromethyl)benzidine (TFMB). The F-sCOP had a large surface area (BET: 346.2 m2 g−1), remarkable enrichment capacity (enrichment factors: 186–782), high selectivity toward fipronil and its metabolites (adsorption efficiency >93.1%), and admirable service life (>60 times). Based on the adsorbent, a novel μ-matrix cartridge extraction-online-μ-solid phase extraction-high performance liquid chromatography-mass spectrometry (μ-MCE-online-μ-SPE-HPLC-MS) method was constructed and used to track trace fipronil and its metabolites in soil. The proposed method exhibited a wide linear range (0.05–1000 ng g−1), low quantitation limits (LOQs: 0.0027–0.011 ng g−1), high recoveries (90.1–119.6%) and good repeatability (RSD ≤10.5%, n = 3) for fipronil analysis. This study paves the way for pesticide analysis in soil risk assessment.

Simultaneous mass spectrometric quantification of trace amines, their precursors and metabolites

ObjectivesTrace amines are powerful neuromodulators influencing the release and reuptake of catecholamines. These low concentrated endogenous amines impact mood, cognition, and hormone regulation. Dysregulation of trace amines have been associated with a variety of diseases, such as schizophrenia, Parkinson’s disease, migraine, depression and more. Succesfull simultaneous quantification of trace amines, their precursors and metabolites would benefit both research and patient care. Since these compounds have various functional groups and are present in biological matrices with large concentration difference, their simultaneous quantification is an analytical challenge. Our goal was to develop a highly sensitive LC-MS/MS assay to simultaneously quantify trace amines, their precursors and metabolites in plasma. MethodsOur method is based on a simple two-step in-matrix derivatization protocol: propionic anhydride (PA) and 3-Ethyl-1-[3-(dimethylamino)propyl]carbodiimide (EDC) in combination with 2,2,2-trifluoroethylamine (TFEA) followed by online solid phase extraction combined with LC-MS/MS. Fifteen metabolites can be measured simultaneously, three precursors, eight trace amines and four metabolites. Validation of this method was performed according to international validation guidelines. The pre-analytical stability of trace amines was assessed. ResultsThis novel method was successful in quantifying trace amines, their precursors, and metabolites in plasma. Using just 50 µl human plasma, we were able to accomplish limit of quantification for 2-phenylethylamine and N-methyl-phenylethylamine of 0.2 nmol/L and 0.1 nmol/L for tyramine and n-methyltyramine. Inter-and intra-assay imprecision was < 15 % for all analytes. Stability assessment showed susceptibility of certain trace amines e.g. 2-phenylethylamine and N-methyl-phenylethylamine to enzymatic degradation in plasma. The addition of the monoamine oxidase inhibitor pargyline to plasma prevented this enzymatic degradation. ConclusionsWe developed a novel LC-MS/MS method that1) uses a new double derivatization technique, 2) is automated with online SPE, 3) uses far less sample volume then previous methods and 4) detects more components in the same sample (eight trace amines, three precursors, and four metabolites) with high specificity and selectivity. Furthermore, addition of MAO A/B inhibitor prevents degradation and guarantees more accurate quantification of trace amines.

Internal multiple interactions-adsorption and external zwitterionic polymer-exclusion of restricted access materials as adsorbent for offline and online extraction of neonicotinoid pesticides in Goji samples

A method based on novel restricted access materials (RAMs) for the determination of neonicotinoid pesticides in Goji samples using offline and online solid phase extraction (SPE) coupled with high-performance liquid chromatography (LC). RAMs were synthesized using poly(chloromethylstyrene-co-divinylbenzene) (PVBC/DVB) microspheres as substrate, styrene (St) and n-vinylpyrrolidone (NVP) were first copolymerized on the interior to construct adsorption sites, and sulfobetaine methacrylate (SBMA) was then polymerized on the exterior to form exclusion sites via two-step surface initiated-atom transfer polymerization. The prepared PVBC/DVB@poly(St-co-NVP)@poly(SBMA) RAMs could efficiently extract neonicotinoid pesticides and automatically exclude proteins. Under the optimized conditions, the developed methods of offline (magnetic SPE and SPE column) and online extraction coupled with LC both using PVBC/DVB@poly(St-co-NVP)@poly(SBMA) RAMs as the extractant, exhibit a wide linearity, low limits of detection and limit of quantification and good inter-day and intra-day precision with satisfactory recoveries. Among these methods, online extraction coupled with LC based on novel RAMs exhibits clear advantages for the determination of neonicotinoid pesticides in Goji samples has clear advantages, such as simple operation by direct injection, short extraction times, and high accuracy with less human error.

Simultaneous determination of 28 illegal drugs in sewage by high throughput online SPE-ISTD-UHPLC-MS/MS

This study developed an online solid-phase extraction ultra-high performance liquid chromatography-tandem mass spectrometry (Online-SPE-UHPLC-MS/MS) method for the analysis of 28 illegal drugs in sewage. To achieve this, 28 isotope internal standards (ISTDs) were added to 3 mL sewage samples, the pH was adjusted to 7–8 using hydrochloric acid or 20% ammonia water, followed by centrifugation, filtration, and analysis using UHPLC-MS/MS. The results indicated an excellent linearity of 1–300 ng L−1, and cotinine in the concentration range of 20–6000 ng L−1, linear correlation coefficient R2 > 0.995, with the limit of detection (LOD) of 0.01–6 ng L−1, and a limit of quantification (LOQ) of 0.1–20 ng L−1. The addition of three concentrates of low (2 ng L−1/40 ng L−1), medium (20 ng L−1/400 ng L−1), and high concentration (200 ng L−1/4000 ng L−1) demonstrated the matrix effect of the target compound between ± 22.0%. The extraction recovery was 70.0–119.4%, and a percent accuracy of 75.7–118.1%. Similarly, the intra- and inter-day precisions were 1.8–20.0% and 1.5–18.9%, respectively. The results cemented the sensitivity, accuracy, reliability, strong specificity, and reproducibility, which can be used to screen 28 illegal drugs in sewage for trace analysis.

Bioanalysis of the Bicycle® toxin conjugate BT5528 and released monomethyl auristatin E via liquid chromatography-tandem mass spectrometry.

Background: The Bicycle® toxin conjugate BT5528 is a novel peptide therapeutic conjugated to the cytotoxic agent monomethyl auristatin E (MMAE). A bioanalytical assay was developed to quantify BT5528 and unconjugated MMAE in human plasma. Methodology: BT5528 quantitation used a protein precipitation procedure followed by LC-MS/MS detection. Quantitation of MMAE required a selective offline and online solid-phase extraction with detection via LC-MS/MS. Results: BT5528 was quantified over the assay range of 5-2500ng/ml and free MMAE was quantified over the assay range of 0.05-50ng/ml. Conclusion: Bioanalytical methods were used in the bioanalysis of intact BT5528 and released MMAE, in a phase I/IIa clinical trial; to date, over 2000 human patient samples have been analyzed.

An Ultra-High-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry Method with Online Solid-Phase Extraction Sample Preparation for the High-Throughput and Sensitive Determination of Ostarine in Human Urine.

Ostarine is frequently misused as a selective androgen receptor modulator (SARM) in sports. Consequently, there is a pressing need for reliable and simple approaches to monitor its presence in biological systems. In this work, we developed a two-dimensional analytical method utilizing online solid-phase extraction (online-SPE) in conjunction with ultra-high-performance liquid chromatography and tandem mass spectrometry (triple quadrupole). This automated 2D separation approach is characterized by minimum manual steps in the sample preparation (only dilute-and-shoot), reflecting high sample throughput and the reliability of analytical data. It provides favorable performance parameters, including a limit of detection of 0.5 pg/mL, high accuracy (relative error = 1.6-7.5%), precision (relative standard deviation = 0.8-4.5%), and sensitivity. Additionally, it demonstrates excellent linearity (r2 = 0.9999) in the calibration range of 0.05 to 25 ng/mL and robustness, with no carryover effects observed. This comparative study revealed a two-decadic-order-lower LOD of the SPE-UHPLC-MS/MS method to the corresponding UHPLC-MS/MS method and the lowest one in the group of currently published LC-MS methods. The World Anti-Doping Agency screening and confirmation criteria were met through the analysis of spiked urine samples from ten healthy volunteers. Accordingly, the proposed method is suitable for routine use in antidoping laboratories.

Thyroid Hormone Metabolites Quantified in Pup and Adult Rat Cerebellum, Cortex and Whole-Brain Samples Using an Automated Online SPE-LC-MS/MS Method.

Changes in thyroid hormone (TH) levels in rat brain at early developmental stages are correlated with adverse effects on offspring development. To characterize the ability of substances to interfere with the TH concentrations in, e.g., rat brain, it is essential to know the mean TH concentrations in this tissue under control conditions. In this publication, an online solid-phase extraction (SPE) liquid chromatography (LC) tandem mass spectrometry (MS/MS) method was validated and used to measure TH metabolites (T4, T3, rT3, T2 and T1) in the brains of untreated rats. Data on TH concentrations in the whole brain and separate data from the cerebellum and the cortex are shown. The corresponding samples were gathered from young rats at postnatal days (PND) 4 and 21/22 and from adult rats. The results show inter alia the high accuracy and precision of the method, and LOQs of 0.02 ng/mL were determined for T1, T2 and rT3 and of 0.15 ng/mL for T3 and T4. Technical variability is low, as shown by the relative standard deviations of 7.5-20%. For our rat model, we found that T4, T3 and T2 concentrations rise from PND4 to PND21, whereas the rT3 concentration decreases; as well as there is no statistical difference between TH concentrations in the male and female rat brain. This method is suitable to analyze TH metabolites in the brain and build up a database of historical TH concentrations in control rats. Together, this yields a robust diagnostic tool to detect potentially adverse disturbances of TH homeostasis in the most vulnerable anatomic structure.

Caffeine quantification in dietary supplements using high-throughput on-line solid phase extraction coupled to Venturi easy ambient sonic-spray ionization mass spectrometry.

Caffeine is present in a large number of beverages and is an additive used in dietary supplements. Therefore, the concern about its quality and safety for consumers has been increasing and hence requires faster and simpler analytical methods to determine the caffeine amount. The high-throughput analysis is an appropriate solution to pharmaceuticals, bioanalysis, forensic and food laboratory routines. In this sense, Venturi easy ambient sonic-spray ionization mass spectrometry (V-EASI-MS), a specific ambient ionization source, is suitable to enable direct analysis of sample solutions in real time and is appropriate to be coupled to liquid chromatography (LC). The development of an on-line solid phase extraction system coupled to V-EASI-MS optimizes the advantages of LC-MS hyphenation by enhancing the figures of merit of the analytical method according to AOAC guidelines and simultaneously minimizing the runtime analysis to 1.5 min per sample, as well as sample preparation steps and solvent consumption, which is currently a challenge for quantitative applications of ambient ionization MS.

An on-line SPE-LC-MS/MS method for quantification of nucleobases and nucleosides present in biological fluids.

Solid phase extraction (SPE) and liquid chromatographic (LC) separation of nucleobases and nucleosides are challenging due to the high hydrophilicity of these compounds. Herein we report a novel on-line SPE-LC-MS/MS method for their quantification after pre-column derivatization with chloroacetaldehyde (CAA). The method proposed is selective and sensitive with limits of detection at the nano-molar level. Analysis of urine and saliva samples by using this method is demonstrated. Adenine, guanine, cytosine, adenosine, guanosine, and cytidine were found in the range from 0.19 (guanosine) to 1.83 μM (cytidine) in urine and from 0.015 (guanosine) to 0.79 μM (adenine) in saliva. Interestingly, methylation of cytidine was found to be significantly different in urine from that in saliva. While 5-hydroxymethylcytidine was detected at a very low level (<0.05 μM) in saliva, it was found to be the most prominent methylated cytidine in urine at a high level of 3.33 μM. Since on-line SPE is deployed, the proposed LC-MS/MS quantitative assay is convenient to carry out and offers good assay accuracy and repeatability.

Development and validation of a bioanalytical method for the quantification of methotrexate from serum and capillary blood using volumetric absorptive microsampling (VAMS) and on-line solid phase extraction (SPE) LC-MS

High-dose methotrexate is part of the polychemotherapy protocols for the treatment of Acute lymphoblastic leukaemia (ALL) with therapeutic drug monitoring (TDM) to adjust leucovorin rescue. An immunoassay is commonly used to analyse serum samples collected via venous blood sampling. However, immunoassays cannot distinguish between the parent drug and its metabolites. Besides, the blood volume required by venous blood sampling is high. Therefore, the aim of this project was to develop a fast, simple, reliable and cost-efficient micro sampling bioanalytical method using capillary blood to minimize the harm of children and to analyse both methotrexate and its metabolites. To achieve this aim, a LC-MS method with on-line solid phase extraction (SPE) for the simultaneous detection of methotrexate and its metabolites from capillary blood using volumetric-absorptive-microsampling (VAMS) technology was developed and fully validated. Besides, the method was also validated and modified for serum samples to compare the results with the immunoassay. A single-quadrupole MS detector was used for detection. Through the use of on-line SPE technology, a lower limit of quantitation of 0.03 µM for MTX and 7-OH-MTX and of 0.05 µM for DAMPA from a 10 μL capillary blood sample was achieved. The accuracy is between 90.0 and 104% and the precision between 4.7 and 12% for methotrexate and its metabolites, respectively. Because of the cross reactivity of the immunoassay a cross-validation was not successful. Besides, a correlation factor of 0.46 for MTX between plasma and whole-blood was found.A fast, simple, reliable and cost-efficient extraction and analysis LC-MS method could be developed and validated, which is applicable in ambulatory and clinical care.

Formation of halogenated forms of bisphenol A (BPA) in water: Resolving isomers with ion mobility – mass spectrometry and the role of halogenation position in cellular toxicity

Halogenated BPA (XBPA) forms resulting from water chlorination can lead to increased toxicity and different biological effects. While previous studies have reported the occurrence of different XBPAs, analytical limitation have hindered the analysis and differentiation of the many potential isomeric forms. Using online solid-phase extraction - liquid chromatography - ion-mobility - high-resolution mass spectrometry (OSPE-LC-IM-HRMS), we demonstrated a rapid analysis method for the analysis of XBPA forms after water chlorination, with a total analysis time of less than 10 min including extraction and concentration and low detection limits (∼5–80 ng/L range). A multi in-vitro bioassay testing approach for the identified products revealed that cytotoxicity and bioenergetics impacts were largely associated with the presence of halogen atoms at positions 2 or 2’ and the overall number of halogens incorporated into the BPA molecule. Different XBPA also showed distinct impacts on oxidative stress, peroxisome proliferator-activated receptor gamma - PPARγ, and inflammatory response. While increased DNA damage was observed for chlorinated water samples (4.14 ± 1.21-fold change), the additive effect of the selected 20 XBPA studied could not explain the increased DNA damage observed, indicating that additional species or synergistic effects might be at play.

Development and validation of online SPE purification coupled to HILIC-fluorescence-MS analysis for the characterization of N-glycans

N-glycans of therapeutic glycoproteins is a critical quality attribute to be addressed. We developed a sensitive method for N-glycan characterization using procainamide (ProcA) labelling and online solid phase extraction (online SPE). N-glycans were enzymatically released, then labeled with ProcA and cleaned up via the online SPE using HILIC chemistry (online HILIC SPE). Two preparation protocols were optimized: a short one (1 h 30) and a long one (18 h). Furthermore, the developed approach was compared to RapiFluor-MS (RFMS) kit (from Waters) and to InstantPC kit (from Agilent) which both include a classical HILIC μElution plate SPE purification. Samples were analyzed using HILIC separation coupled to fluorescence and MS detection (HILIC-FLD-MS) with or without the online HILIC SPE. During the validation, repeatability, intermediate precision, stability, response function and injection volume were tested. Human IgG mix (Multigam®) and NIST mAb standard were used as references as their glycoprofiles are well described. A comparison of three batches of a rituximab biosimilar (Truxima®) and one batch of its originator (MabThera®) was also performed. Online HILIC SPE sample cleanup shows a higher sensitivity and repeatability compared to the classical HILIC μElution SPE. Our online HILIC SPE approach also offers the highest MS signal compared to both commercial kits. However, InstantPC shows the highest FLD signal. The analyses of rituximab samples were in line with the literature showing the efficiency of the method for N-glycan monitoring of biotherapeutics. In conclusion, the results demonstrated the usefulness and ease of application of the developed protocol with the online HILIC SPE purification.

A Novel and Fast Online-SPE-LC-MS/MS Method to Quantify Thyroid Hormone Metabolites in Rat Plasma.

Since the focus in regulatory toxicology has drifted toward the identification of endocrine disruptors, the improvement in determination of alterations in the thyroid hormone system has become more important. THs are involved in several molecular processes important for a proper pre- and postnatal development so that disturbances can inter alia lead to incorrect brain maturation and/or disturbed metabolic processes (thermogenesis or lipolysis). In this publication, a new automated online solid-phase extraction (SPE)-liquid chromatography (LC)-tandem mass spectrometry (MS/MS, xLC-MS/MS) is introduced which simultaneously analyzes total T4, T3, rT3, T2, and T1. Method validation parameters are presented, and the method was positively verified by analyzing control and PTU-treated rat plasma samples (time points day 7, 14, and 28) for their total TH content. The obtained results were compared to published results by using a radioimmunoassay method. The automated SPE system ensures a consistent unified sample preparation, and this method overall showed sufficient specificity and accuracy to detect the given analytes in rat plasma. For the preparation of 50 μL of rat plasma, the following LOQs were established: 0.020 nM for T1, 0.029 nM for T2, 0.023 nM for rT3 and T3, and 3.22 nM for T4. This method is suitable to assess the identification of mechanisms leading to adverse effects, such as disturbed TH metabolism and regulation.

Comprehensive profiling of ceramides in human serum by liquid chromatography coupled to tandem mass spectrometry combining data independent/dependent acquisition modes

Ceramides are sphingolipids with a structural function in the cell membrane and are involved in cell differentiation, proliferation and apoptosis. Recently, these chemical species have been pointed out as potential biomarkers in different diseases, due to their abnormal levels in blood. In this research, we present an overall strategy combining data-independent and dependent acquisitions (DIA and DDA, respectively) for identification, confirmation, and quantitative determination of ceramides in human serum. By application of liquid chromatography–tandem mass spectrometry (LC–MS/MS) method in DIA mode we identified 49 ceramides including d18:1, d18:0, d18:2, d16:1, d17:1 and t18:0 species. Complementary, quantitative determination of ceramides was based on a high-throughput and fully automated method consisting of solid-phase extraction on-line coupled to LC–MS/MS in DDA to improve analytical features avoiding the errors associated to sample processing. Quantitation limits were at pg mL−1 level, the intra-day and between-days variability were below 20 and 25 %, respectively; and the accuracy, expressed as bias, was always within ±25 %. The proposed method was tested with the CORDIOPREV cohort in order to obtain a qualitative and quantitative profiling of ceramides in human serum. This characterization allowed identifying d18:1 ceramides as the most concentrated with 70.8% of total concentration followed by d18:2 and d18:0 with 13.0 % and 8.8 %, respectively. Less concentrated ceramides, d16:1, d17:1 and t18:0, reported a 7.1 % of the total content. Combination of DIA and DDA LC–MS/MS analysis enabled to profile qualitative and quantitatively ceramides in human serum.

Determination of quinclorac and quinclorac methyl ester in honey by online SPE-UPLC-MS/MS

A method employing online solid phase extraction (SPE) coupled to UPLC-MS/MS was developed for the determination of residues of the acid herbicide quinclorac plus its transformation product, quinclorac methyl ester, in honey. The analytical method involved dissolving the honey in a mixture of methanol:water followed by direct injection into a two-dimensional UPLC system which is used to perform an automated SPE cleanup on a reusable phenyl cartridge prior to the target analytes being transferred onto an analytical UPLC column for subsequent chromatographic separation followed by MS/MS detection. The limits of quantitation for quinclorac and quinclorac methyl ester in honey were both set at 0.5 µg kg−1 and the method detection limit was estimated to be 0.012 µg kg−1 for each compound. The working analytical range (0.5–100 µg kg−1) was validated by analysing a series of spiked replicate honey samples. The method was applied to the analysis of various honeys obtained from numerous different commercial sources. Quinclorac was detected in 9 out of 30 samples at concentrations ranging from 0.6 to 31.5 µg kg−1. Quinclorac methyl ester, which is estimated to be significantly more toxic than the parent herbicide itself, was not detected in any honey sample.